Wound monitoring

ABSTRACT

A method of predicting or diagnosing clinical infection of a wound comprising measuring the concentration of a maker associated with an inflammatory response in wound fluid, wherein the maker is a fibronectin fragment, a neutrophil protease or a macrophage protease. Also provided is a use of a wound dressing or biosensor comprising components of an assay system for measuring the concentration of a marker associated with an inflammatory response, wherein the marker is a fibronectin fragment, a neutrophil protease or a macrophage protease, for use in the manufacture of a medicament for predicting the likelihood of clinical infection of the wound or for diagnosing clinical infection of a wound.

The present invention relates to a method of predicting or diagnosingclinical infection of a wound comprising measuring the concentration ofa marker associated with an inflammatory response in wound fluid, forexample the concentration of a neutrophil or macrophage protease. Thepresent invention also relates to devices and kits for use in suchmethods.

In mammals, injury triggers an organised complex cascade of cellular andbiochemical events that result in a healed wound. Wound healing is acomplex dynamic process that results in the restoration of anatomiccontinuity and function; an ideally healed wound is one that hasreturned to normal anatomic structure, function and appearance.

Infection of wounds by bacteria delays the healing process, sincebacteria compete for nutrients and oxygen with macrophages andfibroblasts, whose activity are essential for the healing of the wound.Infection results when bacteria achieve dominance over the systemic andlocal factors of host resistance. Infection is therefore a manifestationof a disturbed host/bacteria equilibrium in favour of the invadingbacteria. This elicits a systemic septic response, and also inhibits themultiple processes involved in wound healing. Lastly, infection canresult in a prolonged inflammatory phase and thus slow healing, or maycause further necrosis of the wound. The granulation phase of thehealing process will begin only after the infection has subsided.

The persistent presence of bacteria in injured tissue results in theprolonged elevation of proinflammatory cytokines such as interleukin-1and tumour necrosis factor alpha (TNF-α). This in turn causes increasesin the levels of matrix metalloproteinases, a decreased level of tissueinhibitors to the metalloproteinases (TIMP), and a decreased productionof growth factors.

Chronically contaminated wounds all contain a tissue bacterial floraThese bacteria may be indigenous to the patient or might be exogenous tothe wound. Closure, or eventual healing of the wound is often based on aphysician's ability to control the level of this bacterial flora.

Current methods used to identify bacterial infection rely mainly onjudgement of the odour and appearance of a wound. With experience, it ispossible to identify an infection in a wound by certain chemical signssuch as redness or pain. Some clinicians take swabs that are thencultured in the laboratory to identify specific organisms, but thistechnique takes time. The prior art also describes the use of certainproteases as an indicator of healing status.

If clinicians could respond to wound infection as early as possible theinfection could be treated topically as opposed to having to useantibiotics. This would also lead to less clinicalintervention/hospitalisation and would reduce the use of antibiotics andother complications of infection.

There is thus a long felt need for a prognostic aid that would assist inpredicting clinical infection of a wound prior to obvious clinicalsymptoms of infection. Such a prognostic aid would allow earlyintervention with suitable treatment (e.g. a topical antimicrobialtreatment) before wound chronicity sets in. There is also a need for adiagnostic aid that would assist in the early diagnosis of clinicalinfection, preferably allowing diagnosis prior to obvious clinicalsymptoms of infection.

It has been discovered that wound fluid from wounds that are apparentlynot clinically infected but which go on to become infected within a fewdays have high levels of neutrophil elastase activity and may also havehigh levels of other inflammatory proteases, such macrophage proteasesand other neutrophil proteases. Similarly, the concentration offibronectin fragments may also provide a useful indication of thelikelihood of subsequent clinical infection; the presence of suchfragments in chronic wounds being mainly due to high levels of elastaseactivity.

According to the present invention, there is provided a method ofpredicting or diagnosing clinical infection of a wound comprisingmeasuring the concentration of a marker associated with an inflammatoryresponse in wound fluid, wherein the marker is a fibronectin fragment, aneutrophil protease or a macrophage protease.

Also provided is a use of a wound dressing or biosensor comprisingcomponents of an assay system for measuring the concentration of amarker associated with an inflammatory response, wherein the marker is afibronectin fragment, a neutrophil protease or a macrophage protease,for use in the manufacture of a medicament for predicting the likelihoodof clinical infection of the wound or for diagnosing clinical infectionof a wound.

Examples of neutrophil/macrophage proteases include elastase, MMP-9,MMP-8, MMP-1, MMP-12 and cathepsin G. Further examples include: collagenpropeptide, a collagen telopeptide, a protease inhibitor, plasmin,lactate dehydrogenase, a cathepsin, a cytokine, a peroxidase enzyme, acortisol free radical or a growth factors. More preferably, the markeris a protease enzyme selected from the group consisting of matrixmetalloproteinases (e.g. MMP-9), neutrophil elastase, plasmin, lowmolecular weight gelatinases and latent or active elastases, interleukinconverting enzymes or tumor necrosis factor (TNFα) converting enzymes.

By “measuring the concentration of a marker associated with aninflammatory response in wound fluid” we include measuring the activityof a marker associated with an inflammatory response in wound fluid. Theterm “wound fluid” refers to any wound exudate or other fluid(preferably substantially not including blood) that is present at thesurface of the wound, or that is removed from the wound surface byaspiration, absorption or washing. The term “wound fluid” does not referto blood or tissue plasma remote from the wound site.

It will be appreciated that the concentration of more than one markermay be measured. In certain embodiments, the concentrations of at leasttwo, three or four markers are monitored.

Measuring the concentration of a marker associated with an inflammatoryresponse in wound fluid allows the likelihood of (or the presence of)clinical infection to be assessed. The step of measuring is preferablycarried out on wound fluid that has been removed from the body of thepatient, but can also be performed on wound fluid in situ.

Any type of wound may be diagnosed for infection according to themethod/use of the present invention. For example, the wound may be anacute wound such as an acute traumatic laceration, perhaps resultingfrom an intentional operative incision, or the wound may be a chronicwound. The method/use of the invention is envisaged as being most usefulin predicting or diagnosing clinical infection of a chronic wound.Preferably, the chronic wound is selected from the group consisting ofvenous ulcers, pressure sores, decubitis ulcers, diabetic ulcers andchronic ulcers of unknown aetiology. Chronic wound fluids inherentlyhave levels of markers such as neutrophil elastase that are many timesthe level found in normal, acute wound fluids. Nevertheless, it has nowbeen found that the levels of such markers are further elevated by asubstantial amount when the chronic wound will become infected.

According to the present invention, the prognostic/diagnostic assay isdesigned so as to provide a correlation between a given concentration ofa marker of an inflammatory response and the likelihood of (or presenceof) clinical infection.

Those skilled in the art will readily be able to determine concentrationlevels of markers of the inflammatory response, which are indicative ofsubsequent progression to clinical infection and/or of the presence ofclinical infection. Preferably, a concentration at least 1.5-, 2-, 2.5-,3.0-, 3.5-, 4.0-, 4.5-, 5.0-, 5.5-, 6.0-, 7.0-, 8.0- or 9.0-fold thebasal level of the marker is considered as begin indicative ofsubsequent progression to clinical infection. By “measuring theconcentration of a marker associated with an inflammatory response” wealso include techniques that produce a positive or negative signal ifthe marker is present at one or more of these concentrations.

Preferably, a concentration at least 1.5-, 2-, 2.5-, 3.0-, 3.5-, 4.0-,4.5-, 5.0-, 5.5-, 6.0-, 7.0-, 8.0-, 9.0-, 10.0-, 11.0-, 12.0-14.0-,16.0-fold the basal level of the marker is considered as beginindicative of the presence of clinical infection. By “measuring theconcentration of a marker associated with an inflammatory response” wealso include techniques that produce a positive or negative signal ifthe marker is present at one or more of these concentrations.

By the “basal level of the marker” we include the level of the markernormally associated with a wound which is not clinically infected andwhich does not subsequently become clinically infected. It will beappreciated that the basal level of the marker may be much higher for achronic wound than for a normal, acute wound.

With regard to the concentration of neutrophil elastase which isassociated with subsequent progression to clinical infection, our dataindicates that an enzyme activity of at least 3,000 mg/min is indicativeof the wound subsequently becoming clinically infected.

As used herein, the term wound fluid is meant to refer to the exudatethat is secreted or discharged by cells in the environment of the wound.This fluid contains cells, both living and dead, and a variety ofinflammatory cytokines.

By the “concentration of a marker of an inflammatory response” is meantthe free concentration of the marker in the wound fluid. Theconcentration of the marker may be assessed in situ, or alternatively asample of wound fluid may be taken as a clinical swab or as a fluidsample.

The concentration of the marker of the inflammatory response may bemeasured by any method known to those of skill in the art. Suitablemethods include those utilising chemical or enzyme-linked reactions, orimmunological (e.g. ELISA, western blots), spectrophotometric,calorimetric, fluorimetric, or radioactive detection based techniques.In one embodiment the concentration of the marker is measured by adip-stick type test. Such a test could be used in the community and bythe patient allowing easier and earlier diagnosis.

To allow measurement of concentration of a marker of the inflammatoryresponse in a wound, a sample of wound fluid must be added to the assaysystem. Measurement may either be made in situ, or fluid may be removedfrom the wound for subsequent analysis. The decision as to which methodis used will depend upon the type of wound in question.

For example, in the case of surface-exposed wounds, a clinical swab,dressing, “dipstick” or other biosensor device may be applied directlyto the surface of the wound. The device should contain the components ofthe assay system for measuring the concentration of the marker so thatthe assay reaction may itself proceed in situ.

The device can then be removed from the wound and the signal measured bythe appropriate means. In many cases, a physician may not actuallyrequire an accurate assessment of the precise concentration of themarker, but may just wish to know whether there is a sufficientconcentration of the marker to warrant prophylactic or curative actionas necessary. In these cases, visible assessment of the dressing may besufficient to allow identification of the specific areas of infection.Unnecessary treatment of healthy granulating tissue can then be avoided.

A dressing that allows mapping of the infected areas of a wound will bepreferable in certain instances. Diagnostic wound mapping sheets thatcould be adapted to the methods of the present invention are describedin GB2323166 (application no. GB 9705081.9), filed on 12 Mar. 1997, theentire content of which is hereby incorporated by reference.

Immobilisation of reaction components onto a dipstick, wound mappingsheet or other solid or gel substrate offers the opportunity ofperforming a more quantitative measurement. For example, in the case ofa reaction linked to the generation of a colour the device may betransferred to a spectrometer. Suitable methods of analysis will beapparent to those of skill in the art.

Immobilisation of the reaction components to a small biosensor devicewill also have the advantage that less of the components (such as enzymeand substrate) are needed. The device will thus be less expensive tomanufacture than a dressing that needs to have a large surface area inorder to allow the mapping of a large wound area.

Methods for the incorporation of the components of the assay reactiononto a clinical dressing, “dipstick”, sheet or other biosensor areroutine in the art. See for example Fägerstam and Karlsson (1994)Immunochemistry, 949-970.

The concentration of the marker may alternatively be measured in anaqueous assay system. Wound fluid may be extracted directly from theenvironment of the wound or can be washed off the wound using a salinebuffer. The resulting solution can then be assayed for the concentrationof the marker in, for example, a test tube or in a microassay plate.

Such a method will be preferable for use in cases in which the wound istoo small or too inaccessible to allow access of a diagnostic devicesuch as a dipstick. This method has the additional advantage that thewound exudate sample may be diluted.

It will be clear that an aqueous assay system is more applicable to usein a laboratory environment, whereas a wound dressing containing thenecessary reaction components will be more suitable for use in ahospital or domestic environment.

Specific embodiments of the present invention will now be described inmore detail, by way of example, with reference to the accompanyingdrawings, in which:—

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 shows the levels of various proteases in diabetic wound fluid.Protease levels including elastase has been corrected for total proteinpresent in the wound fluid sample, thus data is presented as RFU/min/mgprotein (relative fluorescence units per minute−rate of change offluorescence). Patient 8 had 10-fold more elastase activity and withintwo days was hospitalised for severe cellulitis/infection.

EXAMPLES

Clinical Study and Patient Selection

All patients enrolled in this study had diabetic foot ulcers of at least30 days duration and a surface area of at least 1 cm². Patients wereexcluded if the target wound showed any signs of infection or if exposedbone with positive osteomyelitis was observed. Additional exclusioncriteria included concomitant conditions or treatments that may haveinterfered with wound healing and a history of non-compliance that wouldmake it unlikely that a patient would complete the study. Nine patientsmeeting these study criteria were enrolled, and wound fluid collected.Informed consent was obtained from all patients or their authorisedrepresentatives prior to study enrolment and the protocol was approvedby the Ethics Committee at the participating study centre prior to thecommencement of the study.

The study was conducted in accordance with both the Declaration ofHelsinki and Good Clinical Practice.

Protein Assay

Total protein present in each extracted wound fluid sample wasdetermined using the Bradford protein assay. The protein bindingsolution comprises 1 ml Coomassie Brillant Blue stock solution 200mg-Coomassie Brillant Blue G250, Sigma Chemical Co., dissolved in 50 mlethanol-90%); 2 ml orthophosphoric acid (85% w/v); in a final volume of20 ml with distilled water. This solution was filtered (Whatman #1filter paper) and used immediately. The protein level in a sample woundfluid was measured by mixing 10-μl sample or standard with 190-μl of theprotein binding solution in a microtitre well and incubating for 30 minsat ambient temperature prior to reading absorbance at 595 nm. Theconcentration of protein was estimated from a standard calibration ofBSA (bovine serum albumin prepared in distilled water; Sigma ChemicalCo.) ranging from 1.0 to 001 mg/ml.

Protease Activity Assays

The levels of neutrophil-derived elastase, plasmin and matrixmetalloproteinases present in the wound fluid samples were measuredspectrofluorimetrically using substrate activity assays. The substratescomprise short peptides synthesised to mimic the appropriate enzymecleavage site and contain a fluorescent reporter group which is releasedupon hydrolysis. Enzyme activity was determined by measuring the rate ofproduction of the fluorimetric compound, 7-amino 4-methyl coumarin.Activity was expressed either as relative fluorescence units per minute(RFU/min) or change in fluorescence when corrected for total protein(RFU/min/mg protein). Each sample was tested times 6 and the averagevalue calculated. The substrate was prepared at 10 mM-stockconcentration, and diluted to a working concentration of 0.5 mM in theappropriate assay buffer. The reaction mixture, combined in a microtitrewell (black, flat bottomed) comprised 5 μl wound fluid, 175 μl assaybuffer and 20 μl substrate (final concentration 50 μM). The microtitreplate was read immediately at 455 nm (excitation 383 nm) and at timedintervals over the next hour, between readings the plate was covered andincubated at 37° C.

Neutrophil-derived elastase-like activity was estimated using thefluorimetric substrate Methoxy-Alanine-Proline-Valine-7-amino 4-methylcoumarin (Bachem UK, Ltd.) solubilised in methanol. The assay bufferrequired for optimal activity of this enzyme was 0.1M Hepes, pH 7.5containing 0.5M NaCl and 10% dimethyl sulphoxide.

Plasmin-like activity was measured usingMethoxysuccinyl-Alanine-Lysine-Phenylalanine-Lysine 7-amino 4-methylcoumarin (Bachem UK, Ltd.) solubilised in 1 mM-HCl. The assay bufferrequired for plasmin activity was 25 mM Tris-HCl, pH 8.1 containing0.5˜% triton X-100.

Matrix metalloproteinase-like activity was estimated utilising thesubstrate Succinyl-Glycine-Proline-Leucine-Glycine-Proline 7-amino4-methyl coumarin (Bachem, UK, Ltd.) solubilised in methanol. The assaybuffer necessary for maximal MMP activity was 40 mM Tris/HCl, pH 7.4containing 200 mM NaCl and 10 mM CaCl₂.

The results of the assays are shown in FIG. 1. Patient 8 had 10-foldmore elastase activity and within two days was hospitalised for severecellulitis/infection.

The above example has been described for the purpose of illustrationonly. Many other embodiments falling within the scope of theaccompanying claims will be apparent to the skilled reader.

1. A method of predicting or diagnosing clinical infection of a woundcomprising measuring the concentration of a marker associated with aninflammatory response in wound fluid, wherein the marker is afibronectin fragment, a neutrophil protease or a macrophage protease. 2.(canceled)
 3. The method of claim 1 wherein the marker is neutrophilelastase.
 4. The method or use according to claim 1 wherein the wound isa chronic wound.
 5. The method or use according to claim 4 wherein thechronic wound is a chronic ulcer such as a dermal ulcer, venous ulcer,pressure sore or decubitis ulcer.
 6. The method or use according toclaim 1 wherein the concentration of the marker is measured using animmunological, spectrophotometric, colorimetric, fluorimetric, orradioactive detection based technique.
 7. The method or use according toclaim 1 wherein the concentration of the marker is measured by adip-stick type test.